U.S. patent number 6,024,735 [Application Number 08/895,203] was granted by the patent office on 2000-02-15 for process and composition for cleaning soft tissue grafts optionally attached to bone and soft tissue and bone grafts produced thereby.
This patent grant is currently assigned to LifeNet Research Foundation. Invention is credited to Lloyd Wolfinbarger, Jr..
United States Patent |
6,024,735 |
Wolfinbarger, Jr. |
February 15, 2000 |
Process and composition for cleaning soft tissue grafts optionally
attached to bone and soft tissue and bone grafts produced
thereby
Abstract
The invention relates to compositions effective for the
cleansing of mammalian soft tissue optionally attached to bones,
and particularly the removal of blood deposits and bone marrow
therefrom. The compsotions are composed of an aqueous solution
containing as its essential ingredients a detergent having a
functionality of the nature of a polyoxyethylene-23-lauryl either,
a detergent having a functionality of the nature of exyethylated
alkylphenol, and water, where the compositions are free from any
membrane stabilizers. The present invention is also directed to a
method and composition for cleaning cadaveric soft tissue
optionally attached to bone to produce soft tissue grafts
optionally attached to bone suitable for transplantation into a
human. The present method involves removing bone marrow elements,
blood deposits and any bacteria, virus or fungi contamination, from
the donor bone and/or associated soft tissues.
Inventors: |
Wolfinbarger, Jr.; Lloyd
(Norfolk, VA) |
Assignee: |
LifeNet Research Foundation
(Virginia Beach, VA)
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Family
ID: |
27505152 |
Appl.
No.: |
08/895,203 |
Filed: |
July 16, 1997 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
Issue Date |
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619412 |
Mar 21, 1996 |
5820581 |
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620856 |
Mar 20, 1996 |
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646520 |
May 7, 1996 |
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646519 |
May 7, 1996 |
5797871 |
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619412 |
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395113 |
Feb 27, 1995 |
5556379 |
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620856 |
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646520 |
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646519 |
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395113 |
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Current U.S.
Class: |
604/500; 128/898;
623/23.6 |
Current CPC
Class: |
A61B
17/3472 (20130101); A61L 2/0088 (20130101); A61L
2/025 (20130101); C11D 1/83 (20130101); C11D
11/007 (20130101); A61B 2010/0258 (20130101); A61B
2017/00969 (20130101); A61F 2002/2835 (20130101); A61F
2002/4646 (20130101); A61F 2002/4649 (20130101); A61F
2002/4683 (20130101); A61F 2002/4685 (20130101); A61N
7/00 (20130101); C11D 1/29 (20130101); C11D
1/72 (20130101); A61L 2430/34 (20130101); A61B
90/70 (20160201) |
Current International
Class: |
A61B
17/34 (20060101); A61L 2/025 (20060101); A61L
2/02 (20060101); A61L 2/00 (20060101); C11D
11/00 (20060101); C11D 1/83 (20060101); A61F
2/28 (20060101); A61B 19/00 (20060101); A61F
2/46 (20060101); A61B 10/00 (20060101); A61B
17/00 (20060101); A61N 7/00 (20060101); C11D
1/29 (20060101); C11D 1/72 (20060101); C11D
1/02 (20060101); A61M 031/00 () |
Field of
Search: |
;128/898 ;604/28,48,49
;600/36 ;623/16 ;435/1,267,268 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Coggins; Wynn Wood
Assistant Examiner: Blyveis; Deborah
Attorney, Agent or Firm: Hopkins; Susanne
Parent Case Text
REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. patent
application Ser. No. 08/619,412, now U.S. Pat. No. 5,820,581 filed
on Mar. 21, 1996 as a division of U.S. Ser. No. 08/395,113, filed
Feb. 27, 1995, now issued as U.S. Pat. No. 5,556,379; U.S. patent
application Ser. No. 08/620,856 filed on Mar. 20, 1996, which is a
continuation-in-part of U.S. patent application Ser. No.
08/395,113, now issued as U.S. Pat. No. 5,556,379; U.S. patent
application Ser. No. 08/646,520 filed on May 7, 1996, which is a
continuation-in-part of U.S. patent application Ser. No.
08/395,113, now issued as U.S. Pat. No. 5,556,379; U.S. patent
application Ser. No. 08/646,519, now U.S. Pat. No. 5,797,871 filed
on May 7, 1996, which is a continuation-in-part of U.S. patent
application Ser. No. 08/395,113, now issued as U.S. Pat. No.
5,556,379; the disclosures of which are incorporated herein in
their entirety.
Claims
What is claimed is:
1. A method for producing a soft tissue graft suitable for
transplantation into a human, comprising:
sonicating said soft tissue with a solvent comprising one or more
detergents in an ultrasonic cleaner at a temperature and for a time
period effective to produce cleaned soft tissue essentially free
from blood deposits.
2. A soft tissue graft suitable for transplantation into a human,
comprising the cleaned soft tissue produced by the process as
claimed in claim 1.
3. The soft tissue graft of claim 2, wherein said associated soft
tissue comprises one or more members selected from the group
consisting of ligament tissue, tendon tissue, periosteum,
cartilage, menisci and fascia lata tissue.
4. A soft tissue graft produced by the process as claimed in claim
1, wherein said graft is essentially free from bacterial, viral and
fungal contamination.
5. A method for producing a soft tissue graft including attached to
an essentially intact bone suitable for transplantation into a
human, comprising:
inducing a negative pressure mediated flow of a first solvent, said
first solvent comprising one or more detergents, through an opening
in a bone shaft of said essentially intact bone graft and
associated soft tissue to produce a cleaned intact bone graft
including associated soft tissue;
sonicating said essentially intact bone graft and associated soft
tissue in a container with said first solvent using an ultrasonic
cleaner,
wherein said inducing and said sonicating are carried out
simultaneously for a time effective to produce a cleaned intact
bone graft and associated soft tissues essentially free from bone
marrow.
6. A soft tissue graft produced by the process as claimed in claim
5, wherein said graft is essentially free from bacterial, viral and
fungal contamination.
7. A method for producing a soft tissue graft optionally attached
to bone suitable for transplantation into a human, comprising:
sonicating said soft tissue using an ultrasonic cleaning device
with a first solvent comprising one or more detergents to produce a
first cleaned soft tissue graft, and
wherein said first cleaned soft tissue is essentially free from
blood deposits.
8. The method of claim 7, further comprising:
sonicating said first cleaned soft tissue graft using an ultrasonic
cleaning device with a second solvent comprising one or more
members selected from the group consisting of: an antibiotic, an
antimycotic and an antiviral agent, to produce a second cleaned
soft tissue graft; and
sonicating said second cleaned soft tissue graft using an
ultrasonic cleaning device with a third solvent comprising one or
more decontaminating agents to produce a third cleaned soft tissue
graft.
9. The method of claim 8, further comprising:
sonicating said second cleaned soft tissue graft with sterile water
prior to sonication with said third solvent.
10. The method of any one of claims 8 or 9, further comprising:
sonicating said third cleaned soft tissue graft with a fourth
solvent comprising one or more alcohols to produce a fourth cleaned
soft tissue graft.
11. The method of claim 10, further comprising:
washing said fourth cleaned soft tissue graft and with sterile
water.
12. The method of claim 7, further comprising:
agitating said first cleaned soft tissue graft with a second
solvent comprising one or more members selected from the group
consisting of: an antibiotic, an antimycotic and an antiviral
agent, to produce a second cleaned soft tissue graft; and
agitating said second cleaned soft tissue graft with a third
solvent comprising one or more decontaminating agents to produce a
third cleaned soft tissue graft.
13. The method of claim 12, further comprising:
agitating said second cleaned soft tissue graft with sterile water
prior to sonication with said third solvent.
14. The method of claims 12 or 13 further comprising:
agitating said third cleaned soft tissue graft with a fourth
solvent comprising one or more alcohols to produce a fourth cleaned
soft tissue graft.
15. The method of claim 14, further comprising washing said fourth
cleaned soft tissue graft with sterile water.
16. The method of claim 15, wherein said washing comprises one or
more of soaking, sonicating, lavage and agitation.
17. The method of claim 16, wherein said washing is conducted in a
negative pressure environment.
18. The method of any one of claims 1, or 7, wherein said soft
tissue graft comprises one or more members selected from the group
consisting of ligament tissue, tendon tissue, periosteum,
cartilage, menisci and fascia lata tissue.
19. A soft tissue graft produced by the process as claimed in claim
7, wherein said graft is essentially free from bacterial, viral and
fungal contamination.
20. A method for producing a soft tissue graft suitable for
transplantation into a human, comprising:
sonicating said soft tissue in one or more bone cleaning
compositions to produce a cleaned graft;
incubating said cleaned graft in one or more decontaminating agents
to produce a decontaminated graft; and
incubating said decontaminated graft in water to produce soft
tissue graft suitable for transplantation into a human.
21. A soft tissue graft produced by the process as claimed in claim
20, wherein said graft is essentially free from bacterial, viral
and fungal contamination.
22. The method of claim 20, wherein said incubating comprises one
or more or soaking, sonicating, lavage and agitation.
23. The method of claim 22, wherein said washing is conducted in a
negative pressure environment.
24. The method of any one of claims 20 or 22, wherein said bone
cleaning composition comprises:
i) a detergent having a functionality of the nature of a
polyoxyethylene-4-lauryl ether
ii) a detergent having a functionality of the nature of
oxyethytlated alkylphenol, and
iii) water,
wherein said detergent having a functionality of the nature of a
lauryl ether and said detergent having a functionality of the
nature of oxyethylated alkylphenol are present in a weight percent
ration of about 1:2, and wherein said composition does not contain
a membrane stabilizer.
25. A soft tissue graft produced by the process as claimed in claim
24, wherein said graft is essentially free from bacterial, viral
and fungal contamination.
26. A method for producing a soft tissue graft suitable for
transplantation into a human, comprising:
pre-cleaning said graft with water to produce a pre-cleaned
graft;
agitating said pre-cleaned graft in water optionally including one
or more bone cleaning compositions to produced an agitated
graft;
sonicating said agitated graft in one or more bone cleaning
compositions to produce a cleaned graft;
incubating said cleaned graft in one or more decontaminating agents
to produce a decontaminated graft;
wherein said decontaminated graft is suitable for transplantation
into a human.
27. A method for producing a soft tissue graft optionally attached
to bone suitable for transplantation into a human, comprising:
sonicating said soft tissue using an ultrasonic cleaning device
with a first solvent comprising one or more detergents to produce a
first cleaned soft tissue graft, said first cleaned soft tissue
graft us essentially free from blood deposits;
sonicating said first cleaned soft tissue graft using an ultrasonic
cleaning device with a second solvent comprising one or more
members selected from the group consisting of: an antibiotic, an
antimycotic and an antiviral agent, to produce a second cleaned
soft tissue graft; and
sonicating said second cleaned soft tissue graft using an
ultrasonic cleaning device with a third solvent comprising one or
more decontaminating agents to produce a third cleaned soft tissue
graft.
28. A method for producing a soft tissue graft optionally attached
to bone suitable for transplantation into a human, comprising:
sonicating said soft tissue using an ultrasonic cleaning device
with a first solvent comprising one or more detergents to produce a
first cleaned soft tissue graft, said first cleaned soft tissue
graft us essentially free from blood deposits;
agitating said first cleaned soft tissue graft with a second
solvent comprising one or more members selected from the group
consisting of: an antibiotic, an antimycotic and an antiviral
agent, to produce a second cleaned soft tissue graft; and
agitating said second cleaned soft tissue graft with a third
solvent comprising one or more decontaminating agents to produce a
third cleaned soft tissue graft.
Description
FIELD OF THE INVENTION
The subject invention relates to a composition and methods for the
cleansing and disinfection of bones and/or associated attached or
detached soft tissue, and cleansed bone and/or associated soft
tissue produced thereby for use transplantation into a human.
BACKGROUND OF THE INVENTION
A major concern in the area of bone grafting technology is the
effective and safe removal of bone marrow from the less
solvent-accessible cancellous bone spaces within bone grafts.
For bone grafts, human bone may be obtained from cadaveric donors
under sterile conditions in an operating suite environment of local
hospitals. The bone is stored frozen until it is further processed
into small grafts under similar sterile conditions, or under
clean-room conditions. Procurement and processing of human tissues
is typically performed by groups certified by the American
Association of Tissue Banks under standard operating procedures for
the processing of each specific bone graft. For instance, large
bones such as the femur are thawed and debrided of excess tissue
prior to being cut into smaller grafts.
Processing of small bones as well as smaller bone grafts obtained
from large bones includes cleaning of bone marrow from the
cancellous bone spaces using mechanical means, soaking, sonication,
and/or lavage with pulsatile water flow under pressure.
Bone marrow elements include hematopoietic progenitor cells, i.e.,
those stem cells that will ultimately differentiate into red blood
cells, white blood cells, and platelets, among others. These stem
cells are rich in major histocompatibility antigens (i.e., MHC
antigens) that function in immune responses. It is advantageous to
have bone graft material which is essentially free of residual bone
marrow, for use in the preparation of small bone grafts. Large,
essentially whole, bone grafts with minimal residual bone marrow
offer additional advantages in that removal of bone marrow, which
may harbor potential viral particles and/or viral genomes
integrated into the genomes of specific cell types present in the
bone marrow, reduces the potential for transmission of infective
agents such as bacteria and viruses, especially the human
immunodeficiency virus (HIV), since cells capable of harboring the
HIV virus are abundant in bone marrow. The removal of bone marrow
from large or small bone grafts also reduces the bioburden of
viruses which may be present within the bone marrow cells
removed.
Conventional bone-cleaning protocols may include the use of
detergents, alcohol, organic solvents or similar solutes or
combination of solutes designed to facilitate solubilization of the
bone marrow. Common methods may use reduced or elevated
temperatures, for example, between 4.degree. C. to 65.degree.
C.
Ethanol and detergents have been demonstrated to be bacteriocidal
toward certain bacteria, such as gonorrhea, gram negative bacteria,
for example, Yersinia enterocolitica, gram positive bacteria, for
example, Myobacterium tuberculosis and Chlamydia, as well as acid
fast bacteria. Ethanol and detergent solutions also offer
advantages of enhancing solubilization of bone marrow, reducing
surface tension properties of aqueous solutions, and inactivating
viruses and bacteria.
Detergents are amphiphile compounds which facilitate solubilization
of relatively insoluble lipids present in, for example, bone
marrow, yet at high concentrations tend to form micellar structures
(Helenius, A. and Simons, K., "Solubilization of Membranes by
Detergents," Biochim. Biophys. Acta 415:29-79 (1975). The formation
of micellar structures tends to limit the effective concentration
range for detergent solutions, and thus, soaking of bone in a given
volume of detergent solution may not be totally effective in that
the absolute amount of detergent present is limited and if the
amount of lipid material to be solubilized exceeds the
solubilization capability of the detergent present, lipid
solubilization will not be complete. By continually changing the
detergent solution over time, it becomes possible to completely
solubilize all solubilizable lipid present in bone graft.
Typically, hydrogen peroxide is used to oxidize the colored
elements within the bone marrow, which results in a cleaner
appearance. However, such bone often still contains bone marrow
which is extremely immunogenic.
Further, most bone grafts are currently stored in the freeze-dried
state. Freeze-drying removes water from the grafts, but lipid
elements present in the membranes of the bone marrow cells and in
vesicles present in adipocytes (i.e., fat storage cells) typically
leak from the grafts after being placed in their final storage and
distribution containers. These residues often give the appearance
that the graft itself is not clean.
In fact, with conventional bone-cleaning protocols the graft often
harbors bacteria, viruses and/or fungi in the bone marrow. Viruses,
bacteria, and/or fungi may also be present in the soft tissues
associated with bone.
Cleaning of bone marrow from small bone grafts (for example,
tarsels and meta tarsels as small as 1-5 cm) has been described in
the scientific literature and in brochures and documents made
public by groups involved in the procurement and processing of
human tissues. A for-profit public corporation, Cryolife, Inc.
(Marietta, Ga.) promotes a bone cleaning process designated as
VIP.TM. (Viral Inactivation Process) and claims that the process
provides "Cleaner bone through mechanical removal of debris and
tissue such as bone marrow, lipids and blood components" and "Safer
bone through inactivation of pathogens such as HBV and HIV (greater
than 5-log kill) as well as bacteria and fungi" (Cryolife
Orthopedics, Inc., brochure 12, February, 1992; Cryolife literature
directed to Organ and Tissue Procurement Program Directors dated
Feb. 20, 1992).
Minimal information regarding the methods of the process is
available but it is described as a multi-step approach having three
phases: 1) preliminary surface disinfection of procured tissue for
the protection of processing technicians during thawing, debriding
and cutting; 2) cleaning and removal of debris from the cut pieces
with a surfactant at elevated temperature; and 3) terminal
disinfection of the cleaned bone grafts (The Viricidal Capacity of
a Surfactant/Iodophor-Based Viral Inactivation Process for Bone
Allografts, Cryolife documentation). The VIP process is claimed to
both clean bone allografts, e.g., a femur head, and to inactivate a
variety of bacteria and viruses without affecting bone strength or
biological properties. However, according to documents made public
by Cryolife, Inc., the process is used to clean the surfaces of
large bone grafts and to remove bone marrow from the cancellous
bone spaces of small bone grafts cut from the larger grafts.
A second, for-profit publicly held corporation, Osteotech, Inc.,
Shrewbury, N.J., describes a bone graft cleaning process called
Permein ("a combination of ethanol and non-ionic detergent";
Mellonig, J. T., Prewett, A. B., and Moyer, M. P., J. Periodontal
63:979-983 (December, 1992). This Process involves the use of a
solution of ethanol and detergent to clean bone grafts. Details of
the process and detergents utilized are not currently available.
Bone is soaked in the solution and it is claimed that the
combination of ethanol and detergent facilitates permeation of the
solution into bone. The process has been demonstrated to clean
small cut-bone grafts and to be capable of inactivating the HIV in
bone allograft (finely ground bone).
SUMMARY OF INVENTION
The invention addresses the deficiencies and problems in the prior
art by novel compositions which contain a detergent having a
functionality of the nature of a lauryl ether, and a detergent
having a functionality of the nature of oxyethylated alkylphenol,
which are quite effective in removing bone marrow from bones and
bone grafts. The detergents are effective in the formation of
micelles containing bone marrow particles and/or debris. The
concentration of the detergents is such that the bone marrow
particles and/or debris are (1) completely solubilized and (2) kept
in solution. In this fashion, the concentration of the particles
and/or debris is reduced to below the critical micelle
concentration value (CMC). (Critical micelle concentration is a
fixed number, and values are assigned to detergents based on their
respective detergent properties and the molecular weights at which
they function as detergents.) Thus, the particles and/or debris are
in monomeric form so as to be easily washed out of the bone
graft.
The compositions of the invention comprise a superior, safe,
non-toxic, non-pyrogenic solvent and detergent based aqueous agent
that effectively solubilizes and removes bone marrow from bone. The
invention penetrates the less solvent accessible cancerous spaces
within the bone grafts, thus providing effective removal of bone
marrow in one easy and quick cleansing step. Bone grafts cleaned
with the inventive composition retain bone inductive properties
while the bone marrow debris is quickly solubilized and removed.
Bone marrow removal reduces the bioburden of viruses, bacteria and
fungi which grow and may be present in the bone marrow. The
compositions of the present invention also reduce the viral load of
soft tissues associated with, attached to, or removed from bone, to
an undetectable level. These compositions further reduce the
immunogenicity of associated soft tissues.
In addition, the compositions of the invention are easily removed
by a simple washing procedure, and virtually no residual detergents
are present in the bone after washing. The compositions of the
invention are an improvement over the art in providing easy-to-use
excellent cleaning power at a low cost.
Well balanced optimized low concentrations of nonionic and ionic
surfactants and detergents of the invention act synergistically to
lyse, solubilize and keep in solution proteins, lipids, hemopoietic
progenitor cells, red blood cells, white blood cells, platelets and
histocompatible antigens. The surfactants preferably include
Nonoxynol-9, (a known anti-HIV agent), Brij-35 (protein solvent),
Tergitol NP-40 (a lipid solvent) and IGEPAL CA 630. These
surfactants are provided as micelles in optimized critical micelle
concentrations (CMC) to dissolve bone marrow particles and/or
debris, which after being consumed in the cleansing process, are
reduced to a concentration below the CMC value. At that
concentration level the particles and/or debris are in monomeric
form (i.e., act as monomers), and can subsequently be easily
removed via washing steps, leaving no detectable residues in the
bone.
Accordingly, objects of the invention include the development of
compositions which are effective for the cleaning and disinfecting
of bones and/or associated soft tissue, such as by facilitating the
removal of bone marrow and other blood deposits from the
interstitial lumen and cancellous bone space of bone and from soft
tissue.
Another object of the present invention is to provide a bone
cleaning composition which removes most or substantially all of the
bone marrow elements from bone grafts with minimal handling and
processing, to reduce the risk of viral, bacterial and fungal
transmission.
It is a further object of the invention to provide a composition
which improves solvent penetration into and through the bone and/or
associated soft tissue and increases the solubility of bone marrow
and/or blood deposits, facilitating their removal from the
bone.
Another object of the present invention is to provide a method for
cleaning a soft tissue graft optionally attached to bone, by
subjecting the soft tissue to a negative pressure environment.
An object of the present invention is to provide a method for
producing a soft tissue graft optionally attached to bone suitable
for transplant into a human by subjecting the soft tissue
optionally attached to bone to a pressure mediated flow of
solution.
An object of the present invention is to provide a method for
producing a soft tissue graft optionally attached to bone suitable
for transplant into a human by subjecting the soft tissue
optionally attached to bone to a negative pressure mediated flow of
solution.
An object of the present invention is to provide a method for
producing a soft tissue graft optionally attached to bone suitable
for transplant into a human by sonicating the soft tissue
optionally attached to bone with one or more cleaning
solutions.
An object of the present invention is to provide a method for
producing a soft tissue graft optionally attached to bone suitable
for transplant into a human by incubating the soft tissue with on e
or more cleaning solutions.
A yet further object of the invention is to provide methods and
composition for the reduction of the immunogenicity and viral load
of bone and/or associated soft tissues without adversely affecting
the biological and biomechanical properties thereof.
An object of the present invention is to provide a method for
producing a soft tissue graft optionally attached to bone suitable
for transplant into a human by subjecting the soft tissue
optionally attached to bone to a pressure mediated flow of solution
where the solution is recirculated through the bone via the
pressure mediated flow.
These and other objectives of the instant invention have been
realized by use of an aqueous composition which contains as its
essential ingredients a detergent having a functionality of the
nature of a lauryl ether, a detergent having a functionality of the
nature of oxyethylated alkylphenol, and water. The detergent having
a functionality of the nature of a lauryl ether and the detergent
having a functionality of the nature of oxyethylated alkylphenol
should preferably be present in a weight percent ratio of about
3:2:2, respectively.
Preferably, the detergent having a functionality of the nature of
oxyethylated alkylphenol consists of a combination of two compounds
selected from the group consisting of poly(ethylene
glycol)-p-nonyl-phenyl-ether, octylphenol-ethyleneoxide,
polyoxyethylene alcohols, polyethylene glycol
p-isooctylphenylethers, polyoxyethylene nonylphenol and
polyoxyethylene sorbitol esters. Poly(ethylene
glycol)-p-nonyl-phenyl-ether and octylphenol-ethyleneoxide are the
preferred two compounds. The detergent having a functionality of
the nature of a lauryl ether and the two compounds are preferably
present in a weight percent ratio of about 3:1:1, respectively.
In one embodiment, the invention relates to bone cleansing
compositions containing as essential components
i) between about 0.001 to about 2 weight percent (more preferably
from about 0.01 to about 0.5 weight percent and most preferably,
about 0.066 weight percent) of a detergent having a functionality
of the nature of a lauryl ether (such as lauryl ether itself,
preferably polyoxyethylene-4-lauryl ether, and more preferably one
of the Brij series),
ii) between about 0.001 and about 2 weight percent (more preferably
from about 0.01 to about 0.5 weight percent and most preferably
about 0.04 weight percent) of a detergent having a functionality of
the nature of oxyethylated alkylphenol, (such as oxyethylated
alkylphenol itself, preferably poly(ethylene
glycol)-p-nonyl-phenyl-ether and/or octylphenol-ethyleneoxide, and
more preferably Nonoxynol-9 and/or Tergitol NP-40, and/or IGEPAL CA
630), and
iii) water (preferably endotoxin-free deionized/distilled
water).
Preferably, the detergent having a functionality of the nature of
oxyethylated alkylphenol consists of a combination of two compounds
selected from the above-referenced group. The two compounds are
preferably each present in about 0.02 weight percent.
In a preferred embodiment, the invention relates to bone cleansers
composed of an aqueous solution containing as its essential
ingredients
i) about 0.066 weight percent polyoxyethylene-4-lauryl ether,
ii) about 0.02 weight percent poly(ethylene
glycol)-p-nonyl-phenyl-ether (such as, preferably,
Nonoxynol-9),
iii) about 0.02 weight percent octylphenol-ethyleneoxide (such as,
preferably, Tergitol NP-40) or IGEPAL CA 630; and
iv) water (preferably, endotoxin-free deionized/distilled
water).
In another embodiment, the invention also relates to a kit for
cleaning a bone for a bone graft, comprising a solution containing
one or more of the above-described compositions. The kit may
optionally include instructions such as instructions for dilutions
necessary to obtain appropriate weight percentages of the
components in an aqueous solution.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions
The below definitions serve to provide a clear and consistent
understanding of the specification and claims, including the scope
to be given such terms.
Agitation. By the term "agitation" is intended any method of
agitation including mild and vigorous agitation. Agitation may be
carried out in a negative pressure environment.
ALLOWASH.TM. Solution. By the term "ALLOWASH.TM. solution" is
intended those compositions disclosed in co-pending U.S. patent
application Ser. No. 08/620,856 incorporated herein by reference.
Examples of suitable ALLOWASH.TM. compositions include: a cleaning
composition containing essentially about 0.06 weight percent (wt.
%) polyoxyethylene-4-lauryl ether; about 0.02 wt. % poly(ethylene
glycol)-p-nonyl-phenyl-ether; about 0.02 wt. %
octylphenol-ethyleneoxide and endotoxin free deionized/distilled
water.
Blood Deposits. By the term "blood deposits" is intended blood
cells including red blood cells, white blood cells and platelets
and including MHC antigens, and potential viral, bacterial, and
fungal contamination present in soft tissue associated with
bone.
Bone Graft. By the term "Bone Graft" is intended any bone or piece
thereof obtained from a cadaver donor, for example any essentially
intact bone including for example the femur, tibia, ilia, humorous,
radius, ulna, ribs, whole vertebrae, mandibula and/or any bone
which can be retrieved from a donor with minimal cutting of that
bone for example, one half of an ulna, a femur cut in half to yield
a proximal half and a distal half, and/or at least a substantial
portion of a whole bone, i.e. at least one-quarter of a whole bone;
and/or any small cut pieces of bone, for example, iliac crest
wedges, ground bone, Cloward dowels, cancellous cubes, and/or
fibular struts.
Bone Marrow or Bone Marrow Elements. By the term "bone marrow" or
"bone marrow elements" is intended for the purposes of the present
invention the highly cellular hematopoietic connective tissue
filling the medullary cavities and spongy epiphyses of bones which
may harbor bacterial and/or viral particles and/or fungal
particles.
Decontaminating Agent. By the term "decontaminating agent" is
intended one or more agents which remove or inactivate/destroy any
infectious material potentially present in the bone marrow of a
bone graft, for example, such materials including but not limited
to: bacteria, virus, and/or fungi; with such decontaminating agents
including, for example, but not limited to one or more of the
following: an antibacterial agent; an antiviral agent; an
antimycotic agent; an alcohol for example, methyl, ethyl, propyl,
isopropyl, butyl, and/or t-butyl; peracetic acid; trisodium
phosphate; sodium hydroxide; hydrogen peroxide; EXACT.TM. (a
product of ExOxEmis, Inc., Tex.) and/or any detergent.
Detergent. By the term "detergent" is intended any agent which
through a surface action that depends on it possessing both
hydrophilic and hydrophobic properties and/or exerts oil-dissolving
(cleansing) and/or antibacterial and/or antiviral effects, and can
include but is not limited to: anionic detergents, nonionic
detergents, cationic detergents, acridine derivatives, long-chain
aliphatic bases or acids, and the present ALLOWASH.TM. solution.
Examples of specific detergents include: (i) Nonoxynol including
Poly (ethylenegylcol) P-Nonyphenyle ether including
Nonylphenoxypolyethoxyethanol; (ii) Nonidet p.40 including Igepal
CA-630, Antarox A-200 and Triton X-100 (b-octylphenoxyethanol),
including Polyethylene Glycol p-isonctylphenyl Ether including
octylphenoxypolyethoxyethanol and (4-(1,1,3,3-tetramethyl 1
butylphenyl)-00-hydroxy poly (oxy 1,2 ethanediyl); (iii) Brio
including a polyoxyethylene ether and Bru35 including
Polyoxyethylene-23-Lauryl Ether.
Essentially Free From. By the term "essentially free from" is
intended a bone graft where the material removed (i.e., bone
marrow, viral, fungal, and/or bacterial particles) from the bone
graft is not detectable using detection means known in the art at
the time of filing of this application.
Essentially Intact Bone Graft. By the term "essentially intact bone
graft" is intended for the purposes of the present invention any
whole bone including, for example, the femur, tibia, ilia,
humorous, radius, ulna, ribs, whole vertebrae, mandibular, and/or
any bone which can be retrieved from a donor with minimal cutting
of that bone, for example, one half of an ulna, a femur cut in half
to yield a proximal half and a distal half, and/or at least a
substantial portion of a whole bone, i.e., at least one-quarter of
a whole bone.
Mild Agitation. By the term "mild agitation" is intended agitation
achieved through the use of a gyrator shaker or means to achieve a
similar result, including, for example: low pressure pulsatile
lavage wherein induced currents in the solution impact the surface
of bone and associated soft tissue.
Negative Pressure. By the term "negative pressure" is intended for
the purposes of this invention a pressure below atmospheric
pressure, i.e., less than one atmosphere.
Negative Pressure Environment. By the term "negative pressure
environment" is intended a gaseous or liquid environment under
negative pressure including, for example, a negative pressure air
atmosphere, and a liquid environment under negative pressure. A
negative pressure environment in the context of the present
invention means that the soft tissue optionally including bone is,
for example: itself exposed to a negative pressure gaseous
atmosphere or is placed in a container of solution which container
is placed under negative pressure where the tissue is partially or
fully immersed in the solution.
Positive Pressure. By the term "positive pressure" is intended for
the purposes of this invention a pressure at or above one
atmosphere, i.e., greater than or equal to one atmosphere.
Pressure Mediated Flow of Solvent. By the term "pressure mediated
flow of solvent" is intended for the purposes of the present
invention a flow of solvent induced by positive or negative
pressure.
Soft Tissue. By the term "soft tissue" is intended soft tissues
associated with bone regardless of whether they are directly
attached to the bone. Examples of associated soft tissue attached
to bone include, but are not limited to, periosteum, cartilage,
tendons and ligaments. Examples of associated soft tissue not
attached to bone include, but are not limited to, menisci and
fascia lata.
Solvent. By the term "solvent" is intended for the purposes of the
present invention, a liquid cleaning composition capable of:
facilitating the solubilization of lipid, facilitating bone marrow
removal, inactivating viral and/or bacterial particles, and/or
disrupting cell membranes, which may contain, but is not limited
to, one or more of the following: sterile water; saline; a
detergent; an alcohol, for example, ethanol and/or isopropanol,
solvents, a combination of solutes desired to facilitate
solubilization of bone marrow including for example, one or more
of: the present bone cleaning solutions including ALLOWASH.TM.
solution disclosed in co-pending patent application Ser. No.
08/620,856 herein incorporated by reference; a chelating agent; a
virucidal agent; bacteriocidal agent; antimycotic agent; sodium
hydroxide; or similar strong base; organic and/or inorganic acids;
and hydrogen peroxide.
Ultrasonic Cleaner. By the term "ultrasonic cleaner" is intended
any ultrasonic cleaning device capable of operating at: from 20 kHz
to 50 kHz, preferably from about 40 kHz to about 47 kHz, and
includes, for example, Branson ultrasonic cleaner model nos.: 1210,
2210, 3210, 5210 and 8210; or any similar ultrasonic cleaner.
Sonication may be carried out in a negative pressure
environment.
Vigorous Agitation. By the term "vigorous agitation" is intended
agitation achieved through the use of a commercial paint can shaker
or other means which achieve a similar result including, for
example, high pressure pulsatile lavage wherein induced currents in
the solution impact the surface of bone and associated soft
tissue.
II. Cleaning Compositions
The bone cleaning compositions according to the present invention
result in the effective removal of substantially all of the bone
marrow elements within the cancellous bone spaces of bone grafts
optionally having associated soft tissues. The associated soft
tissues include attached soft tissues such as tendons, periosteum,
cartilage and ligaments either attached or removed for cleaning,
and non-attached soft tissues such as fascia lata and menisci. The
bone cleaning composition is effective to remove most or
substantially all of the bone marrow elements from large and small
bone grafts with minimal handling and processing, while reducing
the risk of viral, bacterial and fungal transmission.
The components of the invention should be non-toxic and/or leave a
non-toxic residual concentration of materials in the bone after
flushing with the second solution. In particular, following
cleaning of bone grafts, it is necessary that residual detergents
or other components which may remain associated with the bone graft
are not toxic towards human fibroblast cells expected to migrate
into the bone graft material(s) following implantation.
As explained above, in order that the composition be effective for
the cleansing of bones, and in the removal of bone marrow and like
blood deposits, the composition should be composed of an aqueous
solution containing as its essential ingredients water (such as,
preferably, endotoxin-free deionized/distilled water), and at least
two detergents: a detergent having a functionality of the nature of
a lauryl ether, and a detergent having a functionality of the
nature of oxyethylated alkylphenol/polyethylene glycol phenyl
ether.
The lauryl ether-functioning component should be present at between
about 0.001 to about 2 weight percent, more preferably from about
0.01 to about 0.5 weight percent, and most preferably about 0.066
weight percent. The polyehtylene glycol phenyl ether-functioning
component should be present at between about 0.001 to about 2
weight percent, more preferably from about 0.01 to about 0.5 weight
percent, and most preferably about 0.04 weight percent. In other
words, the lauryl ether-functioning component and the polyethylene
glycol phenyl ether-functioning component are present in a weight
percent ratio of about 1.65:1, respectively.
The lauryl ether-functioning component may itself be a lauryl
ether, which may be selected from the group consisting of
polyoxyethylene-23-lauryl ether (such as Brij series, Lubrol W,
etc.), polyoxyethylene (9) lauryl ether (such as C.sub.12
H.sub.18), polyoxyethylene (9) lauryl ether (such as C.sub.12
H.sub.18), dodecylmaltoside lauryl maltoside (such as dodecyl
.beta.-D-maltopyramoside), decaoxyethylene monolauryl ether (such
as GENAPOL C-100), octaethylene glycolisotridecyl ether (such as
GENAPOL X-080), polyoxyethylene (8) isotridecyl ether (such as
GENAPOL X-080), polyoxyethylene (10) isotridecyl ether (such as
GENAPOL X-100), PEG (10) tridecyl ether (such as GENAPOL X-100),
sodium lauryl sulfate, and sodium dodecyl sulfate.
The lauryl ether-functioning component may be available in a less
concentrated form, such as Brij-35, which is the equivalent of a
35% solution of polyoxyethylene-4-lauryl ether. In such a case, the
weight percent of Brij-35 in the composition of the invention
should be adjusted so that the final weight percent ratio is 0.066
weight percent Brij-35: 0.04 weight percent polyethylene glycol
phenyl ether-functioning component. This is further explained in
the examples below.
The polyethylene glycol phenyl ether-functioning component may
itself be polyethylene glycol phenyl ether, which may be selected
from the group of consisting of poly(ethylene
glycol)-p-nonyl-phenyl-ether, octylphenol-ethyleneoxide,
polyoxyethylene alcohols, polyethylene glycol
p-isooctylphenylethers (such as Triton X series), polyoxyethylene
esters, 1-argitol, polyoxyethylene nonylphenol, and polyoxyethylene
sorbitol esters (such as Tween series and Emasol series).
It is preferable that the polyethylene glycol phenyl
ether-functioning component consists of two compounds selected from
the group consisting of poly(ethylene glycol)-p-nonyl-phenyl-ether,
octylphenol-ethyleneoxide, and polyoxyethylene alcohols,
polyethylene glycol p-isooctylphenylethers, polyoxyethylene
nonylphenol, and polyoxyethylene sorbitol esters. More preferably,
the compounds are poly(ethylene glycol)-p-nonyl-phenyl-ether and
octylphenol-ethyleneoxide. Preferably, the two compounds are each
present in 0.020 weight percent. That is, the lauryl
ether-functioning component and the two compounds are preferably
present in a weight percent ratio of about 3.3:1:1,
respectively.
In a preferred embodiment, the solution includes ALLOWASH.TM.
solution, available from LifeNet Research Foundation, 5809 Ward
Court, Virginia Beach, Va. 23455, ALLOWASH.TM. solution contains
three detergents, i.e., (1) Brij-35 (more specifically,
polyoxyethylene-r-lauryl ether having the chemical formula C.sub.9
H.sub.19 (OCH.sub.2 CH.sub.2).sub.4 OH), (2) Tergitol NP-40
(sometimes known as Nonidet P-40 or NP-40) having the chemical name
octylphenol-ethyleneoxide or IGEPAL.RTM. CA 630, and (3)
Nonoxynol-9 having the chemical name poly(ethylene
glycol)-p-nonyl-phenyl-ether or IGEPAL.RTM. CO 630.
Polyoxyethylene-4-lauryl ether is useful in that it acts as a
protein solubilizing detergent and is used extensively in
electrophoreses of proteins where additional charge problems might
affect separation. Thus, in the cleaning solutions of the
invention, polyoxyethylene-4-lauryl ether is believed to serve in
enhancing solubility of the bulk proteins in the bone marrow,
keeping them "in solution" once solubilized.
Octylphenol-ethyleneoxide and poly(ethylene
glycol)-p-nonyl-phenyl-ether are useful in solubilizing membranes
from cell (plasma) membranes. Thus, in the cleaning solutions of
the invention, octylphenol-ethyleneoxide and poly(ethylene
glycol)-p-nonyl-phenyl-ether are believed to serve primarily in
literally solubilizing the plasma membranes of the bone marrow
cells.
It is important in this invention that at least one of the
detergents be present in a concentration above its critical micelle
concentration. Detergents are typically evaluated based on their
"critical micelle concentration" (CMC). The CMC is that
concentration of detergent in solution where free molecules or
detergent begin to aggregate into micellar structures. In the
cleaning compositions of the invention, the concentration of at
least one of the detergent components should exceed its CMC so that
there is sufficient detergent available in the solution to have
micelles present in the solution to replenish monomeric detergent
as it is consumed in bone marrow solubilization. Notably, however,
the invention is still effective in cleaning bones if the
concentration of one or two of the detergents (especially
octylphenol-ethyleneoxide or Tergitol NP-40) drops below its CMC.
For example, if the cleaning solution becomes diluted.
For Brij-35, the published CMC is approximately 0.092 mM
(millimolar) and was experimentally determined (by detergent
mediated solubilization of an "insoluble" dye) to be about 0.09
mM.+-.0.026 mM; for Nonoxynol 9, the published CMC is approximately
0.0812 mM and was experimentally determined to be about
0.062.+-.0.008 mM; and for Nonidet P-40, the published CMC is
approximately 0.11 to 0.29 mM and was experimentally determined to
be about 0.234.+-.0.005 mM. For this invention, the higher CMC
values reflect greater effectiveness in cleaning bone grafts,
because after the bone marrow particles and/or debris are
"consumed" into micelles their concentration falls below the CMC
values and they are in soluble monomeric form.
The bone cleaning solution can include about 0.0001.times. to
10.times. of a 1.times. detergent solution containing about 0.066
weight percent polyoxyethylene-4-lauryl ether (about 0.066 weight
percent Brij-35), about 0.020 weight percent Tergitol NP-40, and
about 0.020 weight percent Nonoxynol-9 in endotoxin free water
(such as ALLOWASH.TM. solution, where Brij-35 is preferably used).
Preferably, the solution comprises about 0.001.times. to 0.1.times.
of the 1.times. detergent solution, and more preferably, about
0.00.times. to 0.01.times. of the 1.times. detergent solution, and
most preferably, about 0.005.times. to 0.01.times. of the 1.times.
detergent solution.
A 0.01.times. concentration of the ALLOWASH.TM. solution includes a
solution of 1 ml of the 1.times. solution in 99 ml of endotoxin
free water, and other solutions comprise corresponding dilutions
and/or concentrations thereof At a 0.01.times. concentration of
ALLOWASH.TM. solution, all three detergents are above their
critical micelle concentrations (Brij-35 concentration is 0.55 mM,
Non-9 concentration is 0.32 mM, and NP-40 concentration is 0.33
mM).
For example, a 0.01.times. solution includes a solution of 1 ml of
the 1.times. solution in 99 ml of endotoxin free water to provide a
solution comprising 0.00066 weight percent Brij-35, about 0.0002
weight percent Nonidet P-40, and about 0.0002 weight percent
Nonoxynol-9 in endotoxin free water, and a 10.times. solution
comprises 0.66 weight percent Brij-35, about 0.2 weight percent
Nonidet P-40, and about 0.2 weight percent Nonoxynol-9 in endotoxin
free water.
Formulations including solutions of detergents of Brij-35, Nonidet
P-40, and Nonoxynol-9 are disclosed in U.S. patent application Ser.
No. 08/395,113, filed Feb. 27, 1995. U.S. patent application Ser.
No. 07/696,955 discloses these detergents in combination with
membrane stabilizers. Both foregoing applications are hereby
incorporated by reference especially for their disclosure
concerning detergents that are effective in reducing or killing
microorganisms and viruses in a relatively short period of
time.
In accordance with the present invention, the bone cleaning
compositions can include concentrations of about 0.0001.times. to
10.times., preferably 0.001.times. to 0.1.times., more preferably
0.001.times. to 0.01.times. and most preferably 0.005.times. to
0.01.times.. As discussed above, these solutions should preferably
be at a concentration so that upon completion of cleaning of the
bone, e.g., prior to implantation, the concentration of detergents
and/or any of materials in the solution is below a toxic level. For
example, a 0.01.times. solution is a preferred solution, because
removal of 90 percent of this solution from the bone, such as by
subsequent flushing with secondary solutions, reduces the
concentration to approximately a 0.001.times. solution, which is
the non-toxic level. Thus, a 0.01.times. solution provides a highly
cost effective solution having an effective concentration of
detergents without wasting excess detergents.
Optionally, the solution may include alcohols, such as ethanol.
Alcohols are advantageous in that they improve the action of the
cleaning solution of the invention as a bone marrow solubilizing
agent. For instance, ethanol, when included, is included in a
solution of about 5 to 95% ethanol, measured by a volume-to-volume
ratio, and more preferably in the range of about 10 to 30% ethanol,
measured by a volume-to-volume ratio.
The cleaning solutions of the present invention can include any
extraneous components in amounts that are not detrimental to the
cleaning of the bone. For example, components that may be a
detrimental contaminant at higher concentrations can be non-toxic
and/or without consequence to the cleaning efficiency of the
cleaning solution at lower concentrations.
Further, the cleaning solution may include at least one component
selected from the group consisting of antibiotics, antiviral agents
(for example, peroxide generating agents such as EXACT.TM. (e.g.,
trademarked haloperoxidase products marketed by ExOxEmis, Inc., San
Antonio, Tex.)), hydrogen peroxide, permeation enhancers (for
example, fatty acid esters, such as laurate, myristate and stearate
monoesters of polyethylene glycol), organic acids (for example,
citric acid) or dilute solutions of strong acids (for example,
hydrochloric acid).
It is advantageous to clean bones using at least two separate
solutions of the invention. For instance, a first cleaning solution
can include about 0.01.times. of the 1.times. detergent solution.
After the first solution is applied to the bone, a second solution
may be used for flushing the first solution from the bone and for
further reducing bacterial, fungal or viral contaminants.
Preferably, the second solution includes at least one component
selected from the group consisting of endotoxin-free
deionized/distilled water and ethanol. Further, the second solution
may include at least one component selected from the group
consisting of antibiotics, antiviral agents, hydrogen peroxide,
permeation enhancers, organic acids and dilute solutions of strong
acids.
The solutions of the invention are preferably controlled within a
temperature range of 20.degree. C. to 65.degree. C. and maintained
within the temperature range during processing. More preferably,
the temperature range is controlled and maintained at about
27.degree. C. to 55.degree. C. Even more preferably, the
temperature range is controlled and maintained at about 40.degree.
C. to 48.degree. C.
The compositions of the invention are useful with any type or size
of bone and/or attached on unattached associated soft tissue, in
whole or in part. Although the examples below describe use of the
invention on "large" bone grafts, the compositions of the invention
are also useful for the cleaning of any bone or portion of bone,
regardless of size. Further, the examples are directed especially
to human cadaveric bones, but it should be understood that the
invention is equally applicable to bones and soft tissue obtained
from other species.
As used herein, the terms "bone" or "bone graft" may be used
interchangeably, and include whole, intact bones, regardless of
size, or substantial parts of a whole bone with or without
associated soft tissue, or associated soft tissue itself
Practically speaking, bones or bone grafts, as defined herein may
include the range of whole bones down to pieces at least as small
as 1-5 cm. Examples of whole bones include (but are not limited to)
femur, tibia, ilia, humerus, etc., prior to subsequent processing
into specific grafts and may include associated soft tissues.
The composition of the invention is viricidal towards enveloped
viruses such as the HIV, hepatitis, and herpes viruses, for
example. Further viruses which have been killed by the instant
compositions include measles virus, togavirus, enterovirus,
rhinovirus, rubella virus, reovirus, respiratory syncytial virus,
cytomegalovirus, Epstein Barr Virus, Vesicular Stomatitis Virus,
vaccinia virus, rabies virus, influenza virus, parainfluenza virus,
adeno-associated virus, lymphoma virus, human papovirus, and
lymphocytic choriomeningitis virus, for example.
It is possible to monitor the solution exiting the bone cavity to
determine when essentially all of the bone marrow has been removed
from the bone. Monitoring methods include, but are not limited to,
measuring absorbance substantially at 410 nm, and visual monitoring
of the color of the solution exiting the bone.
Other methods of determination of the degree to which the bone
marrow has been removed from the bone graft include: taking core
samples of bone plugs, solubilizing bone marrow in the bone plug
core samples using sodium hydroxide and taking a protein assay of
the same. Visual inspection of the trabecular bone can be examined
using a scanning electron microscope. Gross visual examination can
be performed by cutting the graft open for visual inspection by the
naked eye or with a stereoscope, for example.
III. Soft Tissue Cleaning Processes in Which the Compositions of
the Invention May Be Used
Attached soft tissues (e.g., ligaments, periosteum, cartilage and
tendons) that are attached to bone or removed from the bone; and
soft tissues (fascia lata and menisci) that are associated with but
not attached to bone, can be exposed to detergent solutions
including ALLOWASH.TM. without altering the biomechanical
properties of these soft tissues. Under these conditions, viruses
are inactivated. Cleaning associated soft tissue attached to the
bone, or cleaning the associated soft tissue itself, does not alter
the tissues tensile properties. These soft tissue may be treated
with a viricidal agent. Therefore, soft tissue cleaned of blood
deposits (with or without associated bone) for use in clinical
applications are prepared.
The detergents present in the ALLOWASH.TM. solution are capable of
dissolving membranes of cells and soluble low molecular weight
proteins present in those cells. It can also solubilize low
molecular weight proteins associated with the less soluble
collagens/elastins. Thus, the present invention also includes a
method for producing bone grafts cleaned of bone marrow with
associated soft tissue which soft tissue is not altered in its
tensile properties and which has been treated with a viricidal
agent and is of reduced immunogenicity and has reduced viral
load.
A. Pressure Mediated Flow of Solution
The compositions of the invention may be used in a variety of soft
tissue cleaning procedures where the soft tissue is attached to
bone. For instance, the solutions may be utilized in conventional
flushing procedures to remove blood deposits and bone marrow, which
entails a pressurized flow of solution as a rapidly moving stream
which dislodges blood deposits and bone marrow by impact of the
solvent on the soft tissue and bone graft.
The compositions may also be used in a process such as the
following. Bone materials procured from cadaveric donors, such as
large, essentially whole, bone grafts, are thawed under sterile
conditions at room temperature. The bone is then debrided of excess
cartilaginous tissues on the proximal and distal ends of bones at
their articulation surfaces.
Thereafter, a hole is made substantially midway between the distal
and proximal ends of the bone. The hole may be formed by drilling,
and is preferably formed to have an outside diameter of
approximately 1/4 to 5/8 inch. The hole need only be drilled deep
enough to penetrate the cortical bone to enable a tapping port to
be securely inserted into the hole. After removing as much bone
marrow as possible, a vacuum line is attached securely at one end
to the tapping port after insertion of the tapping port into the
hole.
The opposite end of the vacuum line is securely attached to a
disposable container, via an inlet tube. The inlet tube is
sealingly connected to a disposable container by a stopper means.
The stopper means is preferably a rubber stopper, but any
equivalent, such as, a plastic or cork stopper, may be used to seal
the inlet tube within the disposable container.
An outlet tube of the disposable container is securely attached to
another vacuum line. At the opposite end, the vacuum line is
attached to a vacuum source which is capable of drawing a vacuum in
the range of about 5 inches to 30 inches Hg vapor on the entire
apparatus. The vacuum source used to draw solution through the bone
grafts will be set to draw a vacuum between about 15 and 35 inches
Hg vapor with the preferred range being about 20 and 30 inches Hg
vapor.
After connection of the vacuum apparatus, the bone is immersed into
a solvent containing one of the compositions of the invention,
contained within an open container. The vacuum source is next
turned on to draw the solution through the articulating surfaces
which have been debrided of cartilage as necessary, through the
cancellous bone structure and through the intermedullary canal of
the bone.
As mentioned above, the vacuum drawn is set between about 15 and 35
inches Hg vapor with the preferred range being between about 20 and
30 inches Hg vapor. The actual vacuum level is adjusted such that
the flow rate of solution through the bone graft does not occur so
rapidly that the bone marrow is not effectively solubilized, but
rapidly enough to effectively remove solubilized bone marrow. Flow
rates of solvent should range between about 8 and 32 ml per minute
with the preferred rates being between about 15 and 25 ml per
minute.
The solution enters the bone through the ends at the cartilaginous
surfaces. Restricted flow of the solution through the cartilaginous
ends of the bone minimizes mechanical and/or structural damage to
the cancellous bone by causing a slow flow rate of solvent through
the trabecular bone space occupied by bone marrow. As the solution
is drawn through the bone, it can be collected in a disposable
container. Because the aspirate solution is largely liquid phase,
the solution is deposited into the disposable container as the
vacuum is drawn through the inlet tube end out the outlet tube.
The process may optionally include refilling the container with a
second solution for further processing the bone including flushing
the first solution from the bone. The second solution is drawn
through the cartilaginous articulating surface and then through the
bone cavity and the vacuum line to exit the bone at the opening.
Optionally the second solution may be recirculated through the bone
and associated soft tissue.
The solution is collected in the disposable container and is
initially dark red, reflecting a saturated or nearly saturated
solution of marrow. As the process continues, the solution
gradually turns to a color similar to that of serum as bone marrow
is removed from the graft. By sampling the effluent material being
removed from the bone, such as by a sampling port accessible by use
of a syringe, it is possible to monitor completion of bone marrow
removal by measuring absorbance at 410 to 700 nm. By this method,
it is possible to determine when essentially all of the bone marrow
is removed from the bone graft. Bone marrow solubilization and
cleaning of the bone is essentially complete when eluent protein
concentration reaches a minimal, substantially stable value.
After it has been determined that essentially all of the bone
marrow has been removed from the bone (i.e., the bone graft) the
bone is removed from the container and the solution can be replaced
with a second solution. The bone is then immersed in the second
solution in the container, for further processing. The second
solution to be drawn through the bone graft may include
endotoxin-free deionized/distilled water, ethanolic solutions of
water, or isotonic saline in endotoxin-free deionized/distilled
water. During addition of the second solution to the container, the
vacuum can be shut off until processing of the second solution
through the bone is ready to resume.
The second solution is drawn through the bone in order to reduce
the amount of the first solution in the bone graft and/or to
deliver additional agents to be used in processing of the whole
bone graft. For example, addition of ethanol (50% to 100%, vol to
vol) to reduce bacterial, fungal and/or viral contaminants which
might be present in the bone graft. The use of absolute (100%)
ethanol in the second solution would further serve to dehydrate the
bone, reducing subsequent times needed for freeze-drying.
Since the flow of solution through the bone graft will be less
restricted during flushing with the second solution, the level of
vacuum used (5 to 15 inches mercury vapor) is appropriately reduced
to maintain an appropriate flow rate, preferably between 10 and 15
ml per minute.
The volume of the second solution which is drawn through the bone
varies depending on the size of the bone being processed, the
volume of the intermedullary canal of the bone being processed, and
the concentration of detergent and/or ethanol used in the first
solution, but in general should approximate a volume 10-fold
greater than the volume of the bone graft being processed.
Following completion of flushing of the bone graft with the second
solution, the bone graft may be removed from the container and
processed into smaller bone grafts via procedures previously
established for the production of such grafts.
B. Recirculation Method A: Using Positive and Negative Pressure to
Move Solvent(s) Through an Essentially Intact Bone Graft and
Associated Soft Tissue.
Following thawing under sterile conditions at room temperature, the
bone is prepared for attachment of the solvent line by drilling a
small hole approximately midway between the proximal and distal
ends or by transecting the whole bone approximately midway between
the proximal and distal ends. The bone is attached to the solvent
source and placed into the solvent solution in a sterile cleaning
container. This sterile cleaning container may be the "beaker" of a
Branson ultrasonic cleaner, for example, Models 1210, 2210, 3210,
etc. as size of the bone graft dictates, capable of operating at
between 20 kHz and 50 kHz at temperatures up to 69.+-.5.degree. C.
As solvent solution is forced through the bone graft, it is
collected in a disposable container or recirculated.
The pressure source used to push the solvent solution through the
bone graft will be sufficient to generate a flow of solution from a
pressurized system, preferably a peristaltic pumping system (for
example a Q2V piston size with a V200 Controller from FMI)
approximating up to 2304 milliliters per minute. The use of
peristaltic pumping systems facilitates retention of sterility in
the solutions being induced to flow through the bone grafts. The
actual pressure level or pumping rate is adjusted such that the
flow rate of solution through the bone graft does not occur so
rapidly that the bone marrow is not effectively solubilized, but
rapidly enough to effectively remove solubilized bone marrow. Flow
rates of solvent should range from 180 and 2,304 mls per minute
with the preferred rates being in the range from 500 to 2,000 mls
per minute, most preferably from 1,000 to 1,500 mls per minute.
The first solvent Bone Cleaning Solution or BCS to be drawn using a
negative pressure flow or flushed using a positive pressure flow
through the bone graft can consist of, for example, a sterile
mixture of detergent and/or ethanol or other alcohol, in
endotoxin-free deionized/distilled water. Detergents utilized
include, but are not restricted to, ionic and/or non-ionic
detergents such as polyoxyethylene alcohols (Brij series, Lubrol W,
etc.), polyethylene glycol p-isooctylphenylethers (Triton X
series), Nonidet P-40, nonoxynol-9, polyoxyethylene nonylphenol
(Triton N series, Surfonic N series, Igepal CO series),
polyoxyethylene sorbitol esters (Tween series, Emasol series), the
formulation known as ALLOWASH.TM. Solution (LifeNet Research
Foundation, Virginia Beach, Va., pending patent application Ser.
No. 08/620,856) in concentrations ranging between 0.001 weight
percent to 2.0 weight percent with the preferred concentrations
being in the range of 0.005 to 1.0 weight percent, most preferably
from 0.01 to 0.5 weight percent. The concentration of alcohol which
may be used in the first solution ranges from 5 to 95% (volume to
volume) with the preferred range being from 10 to 30% (volume to
volume). Suitable alcohols include but are not limited to: ethanol,
isopropanol, butanol, n-propanol and methanol.
The second solvent to be drawn using a negative pressure flow or
flushed using a positive pressure flow through the bone graft can
include, for example, hydrogen peroxide in endotoxin-free
deionized/distilled water (for example, from 1 to 5%, preferably 3%
hydrogen peroxide), alcoholic solutions of water, or isotonic
saline in endotoxin-free deionized/distilled water. The second
solvent may be added to the container following removal of the
first solution by simply pouring the second solvent into the
container. During changes of the solution in the container, the
solvent flow should be shut off in order to facilitate solvent
changing in the container. The purpose of the second solvent is to
reduce the amount of the first solvent in the bone graft and/or to
deliver additional agents to be used in processing of the intact
bone graft. For example, addition of hydrogen peroxide (3%),
ethanol, or isopropyl alcohol (50% to 100%, vol to vol) to a
washing solution serves to further reduce bacterial, fungal, and/or
viral contaminants which might be present in the bone graft. The
use of absolute (100%) ethanol or isopropyl alcohol or other
alcohol further serves to dehydrate the bone, thereby reducing
subsequent time needed for freeze-drying. Since the flow of solvent
through the bone graft is less restricted during the flushing with
the second, third, or subsequent solvent(s), the level of pressure
used should be appropriately reduced to maintain an appropriate
flow rate of 1,000 to 1,500 mls per minute. The volumes of the
second, third, etc., solvents may vary depending on the
concentration of detergent and/or alcohol used in the first
solvent, but in general should approximate a volume 10 to 100-fold
greater than the volume of the bone graft being processed.
Following completion of flushing of bone graft with the cleaning
solvents, and washing solvents, the bone graft may be removed from
the sterile container and processed into smaller bone grafts via
procedures previously established for the production of such grafts
or additional solvents may be flushed through the bone graft to add
additional processing procedures/solutions into the total bone
cleaning process.
Optional components may also be added to either the first, second,
third, or subsequent solvents being used to clean and flush,
respectively, the bone graft, including, but not limited to,
antibiotics, antiviral agents (for example peroxide generating
agents such as Exact.TM. a trademarked product marketed by
ExOxEmis, Inc., San Antonio, Tex.), hydrogen peroxide, permeation
enhancers (for example fatty acid esters such as laurate, myristate
and stearate monoesters of polyethylene glycol), organic acids (for
example citric acid) or dilute solutions of strong acids (for
example hydrochloric acid).
(i) Recirculation Method A: In summary
a. Initially about 200 to 1,000 mls, preferably about 400 to 600
mls and most preferably about 500 mls of a first solvent containing
one or more detergents is drawn through the bone graft with
associated soft tissue to waste using a negative pressure mediated
flow of solvent at a temperature of from 37.degree. C. to
44.degree. C.
b. Thereafter the bone and soft tissue is flushed using a positive
pressure mediated flow with a second solvent optionally containing
a detergent (this second solvent may be the same or different from
the first), where the second solvent is flushed to waste or
recirculated through the bone. This flushing or recirculation is
carried out for about 5 to 25 minutes, preferably about 10 to 20
minutes, and most preferably about 15 minutes, or until bone marrow
removal is complete as indicated by absorbance of the effluent
material at 410 nm. (Steps a and b may optionally be repeated using
the same or a different solvent, if necessary, to facilitate
further cleaning. The necessity for further cleaning, as well as
the selection of the particular solvent, can be readily determined
by one of ordinary skill in the art without undue experimentation,
for example, by monitoring absorbance of the effluent material at
410 nm).
c. The second solvent is then removed from the bone graft and soft
tissue by either: (a) flushing to waste using a positive pressure
mediated flow with a third solvent, or (b)(i) drawing according to
step a (about 500 mls) of a third solvent to waste using a negative
pressure flow followed by (b)(ii) flushing using a positive
pressure flow, a third solvent through the bone; where the third
solvent preferably contains a decontaminating agent.
d. A fresh volume of the third solvent is then optionally
recirculated using a positive pressure flow through the bone for a
time period from about 5 to 25 minutes, preferably from about 10 to
20 minutes, and most preferably about 15 minutes.
e. Steps c and d are optionally repeated using new volumes of the
same or different solvents. Preferably, steps c and d are repeated
using a solvent containing one or more antibiotics and/or
antivirals and/or antimycotics followed by removal of the
antibiotics/antivirals/antimycotics, steps c and d may optionally
be repeated using a solvent containing one or more alcohols, and
finally repeated to remove the solvent containing alcohols, using
sterile water as the solvent in steps c and d.
The order of use of solvents and the particular composition of a
particular solvent used in the present process is not critical as
long as the first solvent used is a solvent containing one or more
detergents. The present process includes at least performing steps
a and b using a first solvent containing one or more
detergents.
C. Recirculation Method B: Using Negative Pressure to Pull Solvent
Through a Bone Graft and Associated Soft Tissue
Following thawing under sterile conditions at room temperature, the
bone is prepared for attachment of the solvent line by drilling a
small hole (preferably approximately midway between the proximal
and distal ends of the bone) or by transecting the whole bone
(preferably approximately midway between the proximal and distal
ends of the bone). The bone is attached to the solvent source and
placed into the solvent solution in a sterile cleaning container.
This sterile cleaning container may be the "beaker" of a Branson
ultrasonic cleaner, for example, Models 1210, 2210, 3120, etc., as
size of the bone graft dictates, capable of operating at between 20
kHz and 50 kHz at temperatures up to 69.+-.5.degree. C. As solvent
solution is pulled through the bone graft, it is collected in a
disposable container or recirculated.
The pressure source used to pull solution through the bone grafts
will be sufficient to generate a flow of solution from a system
under negative pressure, preferably a peristaltic pumping system
(for example a Q2V piston size with a V200 Controller from FMI)
approximating up to 2304 milliliters per minute. The use of
peristaltic pumping systems facilitates retention of sterility in
the solutions being induced under negative pressure to flow through
the bone graft. The actual pressure level or pumping rate is
adjusted such that the flow rate of solution through the bone graft
does not occur so rapidly that the bone marrow is not effectively
solubilized, but rapidly enough to effectively remove solubilized
bone marrow. Flow rates of solvent should range from 180 and 2,304
mls per minute with the preferred rates being in the range from 500
to 2,000 mls per minute, most preferably from 1,000 to 1,500 mls
per minute.
The first solvent (Bone Cleaning Solution or BCS) to be pulled
through the bone graft can consist of, for example, a sterile
mixture of detergent and/or ethanol or other alcohol in
endotoxin-free deionized/distilled water. Detergents utilized
include, but are not restricted to, ionic and/or nonionic
detergents such as polyoxyethylene alcohols (Brij series, Lubrol W,
etc.), polyethylene glycol p-isooctylphenylethers (Triton X
series), Nonidet P-40, nonoxynol-9, polyoxyethylene nonylphenol
(Triton N series, Surfonic N series, Igepal CO series),
polyoxyethylene sorbitol esters (Tween series, Emasol series), the
formulation known as ALLOWASH.TM. Solution (LifeNet Research
Foundation, Virginia Beach, Va., pending patent application Ser.
No. 08/620,856) in concentrations ranging between 0.001 weight
percent to 2.0 weight percent with the preferred concentrations
being in the range of 0.005 to 1.0 weight percent, most preferably
from 0.01 to 0.5 weight percent. The concentration of alcohol which
may be used in the first solution ranges from 5 to 95% (volume to
volume) with the preferred range being from 10 to 30% (volume to
volume). Suitable alcohols include but are not limited to:
methanol, ethanol, propanol, isopropanol, n-propanol, and/or
butanol.
The second solvent to be drawn through the bone graft can include,
for example, hydrogen peroxide in endotoxin-free
deionized/distilled water (for example, from 1 to 5%, preferably 3%
hydrogen peroxide), alcoholic solutions of water, or isotonic
saline in endotoxin-free deionized/distilled water. The second
solution may be added to the container following removal of the
first solution by simply pouring the second solution into the
container. During changes of the solution in the container, the
solvent flow should be shut off in order to facilitate solvent
changing in the container. The purpose of the second solution is to
reduce the amount of the first solution in the bone graft and/or to
deliver additional agents to be used in processing of the whole
bone graft. For example, addition of hydrogen peroxide (3%),
ethanol, or isopropyl alcohol (50% to 100%, volume to volume) to a
washing solution would serve to further reduce bacterial, fungal,
and/or viral contaminants which might be present in the bone graft.
The use of absolute (100%) ethanol or isopropyl or other alcohol
further serves to dehydrate the bone, thereby reducing subsequent
time needed for freeze-drying. Since the negative pressure induced
flow of solution through the bone graft is less restricted during
the use of the second, third, or subsequent solvent(s), the level
of pressure used should be appropriately adjusted to maintain an
appropriate flow rate of 1,000 to 1,500 mls per minute. The volumes
of the second, third, or subsequent solvent(s) may vary depending
on the concentration of detergent and/or ethanol or other alcohol
used in the first solution, but in general should approximate a
volume 10 to 100-fold greater than the volume of the bone graft
being processed.
Following completion of negative pressure mediated drawing of the
bone graft with the cleaning and washing solvents, the bone graft
may be removed from the sterile container and processed into
smaller bone grafts via procedures previously established for the
production of such grafts or additional solutions may be flushed
through the bone graft to add additional processing
procedures/solvents into the total bone cleaning process.
Optional components may also be added to either the first, second,
third, or subsequent solvents being used including, but not limited
to, antimycotics, antibiotics, antiviral agents (for example
peroxide generating agents such as Exact.TM. a trademarked product
marketed by ExOxEmis, Inc., San Antonio, Tex.), hydrogen peroxide,
permeation enhancers (for example fatty acid esters such as
laurate, myristate and stearate monoesters of polyethylene glycol),
organic acids (for example citric acid) or dilute solutions of
strong acids (for example, hydrochloric acid).
(i). Recirculation Method B: In summary
a. Initially about 200 to 1,000 mls, preferably about 400 to 600
mls and most preferably about 500 mls of a first solvent containing
one or more detergents is drawn through the bone graft to waste
using a negative pressure mediated flow of solvent at 37.degree. C.
to 44.degree. C.
b. Thereafter a second solvent which may be the same or different
from the first is then: (a) drawn to waste or (b) recirculated
through the bone using a negative pressure medicated flow of
solvent. This drawing or recirculation is carried out for about 5
to 25 minutes, preferably about 10 to 20 minutes, and most
preferably about 15 minutes, or until bone marrow removal is
complete as indicated by absorbance of the effluent material at 410
nm. (Steps a and b may optionally be repeated using the same or a
different solvent, if necessary, to facilitate further cleaning.
The necessity for further cleaning, as well as the selection of the
particular solvent, can be readily determined by one of ordinary
skill in the art without undue experimentation, for example, by
monitoring absorbance of the effluent material at 410 nm).
c. The second solvent is then removed from the bone graft by
drawing a third solvent according to step a (about 500 mls) to
waste using a negative pressure flow. The third solvent preferably
contains a decontaminating agent.
d. A fresh volume of the third solvent is then optionally drawn or
recirculated through the bone for a time period of from about 5 to
25 minutes, preferably from about 10 to 20 minutes, and most
preferably about 15 minutes using a negative pressure redirected
flow of solvent.
e. Steps c and d are optionally repeated using new volumes of the
same or different solvents. Preferably, steps c and d are repeated
using a solvent containing one or more antibiotics and/or
antivirals and/or antimycotics followed by removal of the
antibiotics/antivirals/antimycotics, steps c and d may optionally
be repeated using a solvent containing one or more alcohols, and
finally repeated to remove the solvent containing alcohols, using
sterile water as the solvent in steps c and d.
The order of use of solvents and the particular composition of a
particular solvent used in the present process is not critical as
long as the first solvent used is a solvent containing one or more
detergents. The present process includes at least performing steps
a and b using a first solvent containing one or more
detergents.
D. Sonication
An embodiment of the present invention involves a process for
cleaning essentially intact bone grafts including attached soft
tissue. First, bone materials procured from cadaveric donors are
thawed. The bone is optionally debrided of external soft tissues.
This debridement can include removal of excess cartilaginous
tissues on the proximal and distal ends of bones at their points of
articulation. The bone and attached associated soft tissue is
attached to the vacuum source and placed into the solvent solution
in an appropriate container, for example, a sterile basin or the
tank of a commercially available ultrasonic cleaner, for example,
Branson models 1210, 2210, 3210, 5210, or 8210, each of which hold
essentially larger and larger volumes of cleaning solution.
Preferably, the ultrasonic cleaner operates at (at) least 20 kHz,
more preferably 30 kHz to 50 kHz, and most preferably 40 kHz to 47
kHz. The container is closed around the vacuum line or point of
attachment to the bone graft to restrict movement of cleaning
solution and a vacuum is applied to the system. The ultrasonic
cleaner is then turned on with confirmation of cavitation
performed. As solvent solution is drawn through the bone graft, it
is collected in the disposable container. Solutions in the
container can be changed by addition of new solution through a
filling port. Co-pending application Ser. No. 08/646,520, filed May
7, 1996, entitled "A Recirculation Method for Cleaning Essentially
Intact Bone Grafts Using Pressure Mediated Flow of Solvents and
Bone Grafts Produced Thereby" is incorporated herein in its
entirety and describes in detail methods for cleaning essentially
intact bone grafts.
The vacuum source used to draw solution through the bone grafts
will be between 15 and 35 inches Hg with the preferred range being
between 20 and 30 inches Hg. The actual vacuum level is adjusted
such that the flow rate of solution through the bone graft does not
occur so rapidly that the bone marrow is not effectively
solubilized, but rapidly enough to effectively remove solubilized
bone marrow. Flow rates of solvent should range between 8 and 32
mls per minute with the preferred rates being between 15 and 25 mls
per minute.
The first solvent to be drawn through the bone graft will consist
of a sterile mixture of detergent and/or alcohol, for example,
ethanol or isopropanol in endotoxin-free deionized/distilled water.
Detergents utilized may include, but not be restricted to, ionic
and/or nonionic detergents such as polyoxyethylene alcohols (Brij
series, Lubrol W, etc.), polyethylene glycol p-isooctylphenylethers
(Triton X series), Nonidet P40/Igepal CA 630, Nonoxynol-9 Igepal CO
630, polyoxyethylene nonylphenol (Triton N series, Surfonic N
series, Igepal CO series), polyoxyethylene sorbitol esters (Tween
series, Emasol series), the formulation known as ALLOWASH.TM.
Solution (LifeNet Research Foundation, Virginia Beach, Va.) in
concentrations ranging between 0.001 wt % to 2 wt % with the
preferred concentrations being between 0.01 wt % and 0.5 wt %. The
concentration of alcohol which may be used in the first solution
ranges between 5% and 95% (volume to volume) with the preferred
range being between 10% and 30% (volume to volume).
The second solvent to be drawn through the bone graft will consist
of endotoxin-free deionized/distilled water, alcoholic solutions of
water, or isotonic saline in endotoxin-free deionized/distilled
water. The second solution may be added to the container, used to
contain the first solution, using the filling port. During addition
of the second solvent to the container, the vacuum should be shut
off in order to facilitate filling of the container. Ultrasonic
cleaning may be used during this second solvent process, however it
is generally not necessary since the first solvent processing is
maximally effective in facilitating removal of bone marrow and bone
marrow elements. The purpose of the second solvent is to reduce the
amount of the first solution in the bone graft and/or to deliver
additional agents to be used in processing of the essentially
intact bone graft. For example, addition of ethanol or isopropanol
(50% to 100%, volume to volume) to the washing solvent would serve
to reduce bacterial, fungal, and/or viral contaminants which might
be present in the bone graft. The use of absolute (100%) ethanol in
the second solution would further serve to dehydrate the bone,
reducing subsequent times needed for freeze-drying. Since the flow
of solvent through the bone graft will be less restricted during
the flushing with the second solvent, the level of vacuum used
should be appropriately reduced to maintain an appropriate flow
rate between 10 and 15 ml per minute. The volume of the second
solvent may vary depending on the concentration of detergent and/or
ethanol used in the first solvent, but in general should
approximate a volume 10-fold greater than the volume of the bone
graft being processed.
Following completion of flushing of bone graft with the second
solution, the bone graft may be removed from the sterile container
and optionally processed into smaller bone grafts via procedures
previously established for the production of such grafts.
Optional components may also be added to either the first or second
solvent being used to clean and flush, respectively, the bone
graft, including but not limited to, antibiotics, antiviral agents
(for example, peroxide generating agents such as Exact (a
trademarked product marketed by ExOxEmis, Inc., San Antonio,
Tex.)), hydrogen peroxide, permeation enhancers (for example, fatty
acid esters such as laurate, myristate and stearate monoesters of
polyethylene glycol), organic acids (for example, citric acid) or
dilute solutions of strong acids (for example, hydrochloric
acid).
Soft tissue not attached to bone can also be processed as above
without the use of a pressure mediated flow of solution.
E. Preferred Method for Cleaning of Non-Bone Tissues.
The soft tissue graft is cleansed using a pulsatile water apparatus
making sure to remove as much of the marrow elements and blood
deposits as possible. Preferably soft tissue grafts attached to
bone are then placed in a sterile can entirely filled with sterile
water (approximately 3 liters of sterile water) at 27.degree. C. to
44.degree. C., preferably 40.degree. C. Alternatively, the present
cleaning solution can be used in place of sterile water or an
additional agitation can be preformed using the present cleaning
solution after agitation using water. Both Achilles tendons and
patellar ligaments from the same donor may be placed in the same
can.
The can is then agitated for 10-12 minutes at 450-550 rpms
preferably for at least 12 minutes at 500 rpm's. The graft is then
visually inspected and if is not cleaned sufficiently, it is again
cleaned using for example pulsatile lavage, followed by further
cleaning using vigorous agitation. Such determination can be
readily made by one of ordinary skill in the art to which the
present invention pertains.
For soft tissue grafts attached to bone and soft tissue grafts not
attached to bone, the graft is then placed in a basin containing a
1:100 dilution of the present composition (Bone Cleaning Solution
(BCS)) or other surfactant(s) for at least 15 minutes, preferably
15 minutes. The tissue can be incubated by soaking, sonicating or
agitation, optionally in a negative pressure environment at a
pressure from about 20 to 30 inches of mercury vapor, preferably by
soaking at atmospheric pressure. The graft is then rinsed at least
three times, preferably 3 times with sterile water to remove any
residual surfactants. The sterile rinse water which accumulates in
the "rinse" basin is then discarded.
The entire fashioned graft is then placed in a small basin
containing one or more decontaminating agents, preferably U.S.P.
grade 70% isopropyl alcohol for 2-5 minutes. The graft is then
placed in a sterile basin containing an antibiotic solution. The
fashioned graft remains in this solution for at least 15 minutes,
preferably 15 minutes. The graft should not be left in the
isopropyl alcohol for more than 5 minutes, as this will tend to
desiccate the soft tissue.
The graft is then thoroughly soaked by immersing each deposit into
a sterile water bath for a minimum of five minutes, preferably 15
minutes, to remove any remaining reagents. Using the pulsative
water apparatus, any remaining marrow elements and/or blood
deposits are removed from the bone and/or soft tissue. If the
graft(s) are to be freeze dried they are placed on sterile fine
mesh gauze. The gauze is trimmed just beyond the edges of the
graft.
The graft(s) are then measured, assigned identification numbers,
and packaged as appropriate.
(i) Summary of Soft Tissue Cleaning Protocols:
Associated soft tissue is cleaned according to the present
invention as follows:
a. The graft is incubated in one or more of the present bone
cleaning solutions with the incubation including one or more of
immersion, soaking, agitation (e.g., gyratory shaker or paint can
shaker) and sonication optionally in a negative pressure
environment, to produce a bone cleaning solution cleaned graft;
b. the bone cleaning solution cleaned graft is then rinsed with
water optionally including one or more decontaminating agents to
produce a rinsed graft;
c. the rinsed graft is incubated in one or more decontaminating
agents with the incubation including one or more of immersion,
soaking, agitation (e.g., gyratory shaker or paint can shaker) and
sonication optionally in a negative pressure environment, to
produce a decontaminated graft; and
d. the decontaminated graft is incubated in water optionally
including one or more decontaminating agents, optionally in a
negative pressure environment, to produce a cleaned graft suitable
for transplantation into a human.
Prior to step (a), the graft may optionally be subjected to:
pre-cleaning with water optionally including one or more of the
present bone cleaning solutions, pre-cleaning including cleaning by
one or more of pulsatile lavage, soaking, immersion, agitation and
sonication, optionally in a negative pressure environment, to
produce a pre-cleaned graft, and
agitating said pre-cleaned graft in water optionally including one
or more of the present bone cleaning solutions, agitating including
agitation achieved by using a gyratory shaker or a paint can
shaker, optionally carried out in a negative pressure
environment.
After step (c) and prior to step (d) the graft may optionally be
incubated in one or more decontaminating agents including one or
more antibiotics, incubation including one or more of immersion,
soaking, agitation (e.g., gyratory shaker or paint can shaker) and
sonication optionally carried out in a negative pressure
environment, to produce an antibiotic cleaned graft .
EXAMPLES
The following illustrative examples describe the instant invention
in more detail. However, they are not intended to limit the scope
of the specification and claims.
Example I
A femur was thawed, debrided of excess soft tissue (including the
excess cartilage present on the articulating surfaces) and a hole
approximately 1/4 to 5/8 inch outside diameter was drilled in the
bone shaft approximately midway between the distal and proximal
ends of the bone. The hole was only drilled deep enough to
penetrate the cortical bone so that intramedullary bone marrow
could be flushed from the bone and so a tapping port could be
securely inserted into the hole. The vacuum line was attached
securely to the tapping port.
Two liters of a solution of 10% ethanol in a 0.01.times. solution
containing 0.0066 weight percent Brij-35, 0.002 weight percent
Nonidet P-40, and 0.002 weight percent Nonoxynol-9 in endotoxin
free water were added to an open container in a clean room
environment under sterile conditions. The femur having the vacuum
line attached via the tapping port was then placed into the
container, and immersed towards the bottom of the container.
The temperature of the cleaning solution was adjusted to 45.degree.
C. prior to addition of the bone graft. A vacuum was applied to the
system and maintained in the range of 25 to 27 inches Hg vapor. The
flow rate of solution through the bone graft was maintained at
approximately 10 ml per minute by adjusting the vacuum. The
solution collected in the disposable container was dark red
initially, turning to a color similar to that of serum as bone
marrow was removed from the graft. By sampling the effluent
material being removed from the bone graft, via a sampling port
accessible by use of a syringe, it was possible to monitor
completion of bone marrow removal by measuring absorbance at 410
nm, to determine when essentially all of the bone marrow was
removed from the bone graft. After drawing two liters of first
solution through the bone graft, the vacuum to the system was
discontinued and the open container was refilled with one liter of
endotoxin-free deionized/distilled water. The vacuum was reapplied
to the system. The deionized/distilled water was flushed through
the bone graft at approximately 15 ml per minute to remove the
detergent solution. Following the flushing of detergent solution
from the bone graft, vacuum was discontinued to the system and the
bone graft was removed from the open container, after which the
vacuum line and tapping port were removed. The bone graft was then
ready for further processing into small bone grafts as
required.
Example II
A femur was thawed, prepared and cleaned in the same manner as
indicated in Example I, with the following exceptions. The bone was
cut in half using a bone saw. The proximal end of the femur was
used in this example, however, the distal end of the femur would be
similarly processed. Pulsavac lavage was applied to remove bone
marrow from the luminal space. One liter of solution of 10% ethanol
in a 0.01.times. solution container 0.0066 weight percent Brij-35
(0.55 mM), 0.002 weight percent Nonidet P-40 (0.33 mM), and 0.002
weight percent Nonoxynol-9 (0.32 mM) in endotoxin free water was
added. A sealing cap was placed over the cut end of the bone graft
and secured using a clamping device. A vacuum line was attached
securely to an access line in the sealing cap.
The bone graft having the vacuum line attached via the sealing cap
and access line was then placed into the container, and immersed
towards the bottom of the container. The temperature of the
cleaning solution was adjusted to room temperature (approximately
27.degree. C.) prior to addition of the bone graft. Vacuum was
applied to the system and maintained in the range of about 25 to 27
inches Hg vapor. The flow rate of solution through the bone graft
was maintained in the range of about 25 to 27 inches Hg vapor. The
flow rate of solution through the bone graft was maintained at
approximately 10 ml per minute by adjusting the vacuum.
The solution collected in the disposable container was initially
dark red, and turned to a color similar to that of serum as bone
marrow was removed from the graft. By sampling the effluent
material being removed from the bone graft, via a sampling port
accessible by use of a syringe, it was possible to monitor
completion of bone marrow removal by monitoring absorbance at 410
nm, and it was possible to determine when essentially all of the
bone marrow was removed from the bone graft.
After drawing one liter of first solution through the bone graft,
the vacuum to the system was discontinued and the open container
was refilled with one liter of endotoxin-free deionized/distilled
water (second solution), after which vacuum was reapplied to the
system. the deionized/distilled water was flushed through the bone
graft at approximately 15 ml per minute to remove the detergent
solution. Following the flushing of detergent solution from the
bone graft, vacuum was discontinued to the system and the bone
graft was removed from the container. Next, the sealing cap and
vacuum line were removed. The bone graft was then ready for further
processing into small bone grafts as required.
Example III
Achilles Tendon
Using a band or Stryker saw, jagged edges were cut from a calcaneus
bone block left from the recovery procedure. The bone block was at
least 2.0 cm long (proximal to distal measurement). Any large
amounts of muscle were removed from the tendon material. Leaving as
much soft tissue as possible on the graft initially protects the
tendon during the shaking phase. This prevents the calcaneus from
disrupting the tendon fibers during agitation.
The Achilles tendon graft was cleansed using a pulsatile water
apparatus making sure to remove as much of the marrow elements as
possible. The graft was then placed in a sterile can entirely
filled with sterile water (approximately 3 liters of sterile water)
at 40.degree. C. The can was then placed in a sterile polyethylene
bag and agitated for 12 minutes at 500 rpm's. The graft was then
visually inspected.
The sterile water was then decanted and any remaining gastrocnemius
muscle and fat from the superior aspect of the graft was removed
with care being taken not to remove any of the tendon itself The
graft was then placed in a basin containing a 1:100 dilution of
ALLOWASH.TM. solution. The graft was then rinsed three times with
sterile water to remove any residual surfactants. The sterile rinse
water which accumulated in the "rinse" basin was then
discarded.
The entire fashioned graft was then placed in a small basin
containing U.S.P. grade 70% isopropyl alcohol for 5 minutes. The
graft was then placed in a sterile basin containing an antibiotic
solution. The fashioned graft remained in this solution for 15
minutes.
The graft was then thoroughly soaked by immersing into a sterile
water bath for 15 minutes, to remove any remaining reagents. Using
the pulsative water apparatus, any remaining marrow elements were
removed from the bone. The graft was placed on sterile fine mesh
gauze. The gauze was trimmed just beyond the edges of the
graft.
The length of the graft was measured to the nearest tenth of a
centimeter. The graft was assigned identification numbers.
The graft and gauze were rolled into a tube and together placed
into a 250 ml glass bottle and labeled as appropriate.
TABLE I
__________________________________________________________________________
Achilles Tendon Treatment Tensile Force (N) Strain (in/in) Tensile
Strength (MPa) Youngs Modulus (MPa)
__________________________________________________________________________
BCS 318.9 0.45 8.828 59.332 BCS 988.395 0.51 1.895 11.487 BCS
375.43 0.59 4.773 27.849 BCS 1133.407 0.64 2.857 20.083 BCS 614.744
0.5 0.439 2.825 NBCS 498.646 0.27 8.874 70.873 NBCS 937.685 0.62
4.607 9.724 NBCS 897.651 0.44 3.938 9.515 NBCS 1293.988 0.64 0.327
6.811 NBCS 853.169 0.39 0.609 7.827 Average BCS 686.18 0.54 3.7584
24.315 SD 363.29 Max/Min 1133/319 Range 814.51 Average NBCS 896.23
0.47 3.671 20.950 SD 282.78 Max/Min 1294/499 Range 795.34
__________________________________________________________________________
BCS = Bone Cleaning Solution (0.01X ALLOWASH Solution) MPa =
MegaPascals NBCS = No Bone Cleaning Solution (EndotoxinFree
Deionized/Distilled Water (N) = Newtons Strain units are
dimensionless
Example IV
Patellar Ligament
The patella with patellar ligament intact was removed from the
tibia by fashioning a section of bone from the tibial tuberosity
with a band saw or Stryker saw. This bone block was at least 2.8 cm
long (2.8 cm distal to the patellar ligament insertion site), at
least 2.0 cm wide, and at least 1.2 cm thick (anterior-posterior
measurement). Both the medial and lateral sides of the patella were
trimmed to expose the marrow. Any large amount of muscle was
removed from the tendon material. Note that leaving soft tissue on
the graft initially protects the tendon during the shaking phase.
This soft tissue prevents the patella and tibia bone blocks from
later disrupting the tendon fibers during agitation.
The patellar ligament was cleansed using a pulsatile water
apparatus making sure to remove as much of the marrow elements as
possible. The graft was then placed in a sterile can entirely
filled with sterile water at 40.degree. C. This required
approximately 3 liters of sterile water. Note that both achilles
tendons and patellar ligaments may be placed in the same shaker
can. However, achilles tendons and patellar ligaments should not be
placed with other cut grafts from the same donor in that other cut
grafts can cut and tear soft tissue grafts. The can was then placed
in a sterile polyethylene bag and agitated for 12 minutes at 500
rpms.
After decanting the sterile water solution from the can, all
extraneous soft tissue, including retinaculum, were carefully
removed using sharp and blunt dissection techniques. Care was taken
not to cut the ligament fibers during dissection.
The patellar ligament was greater than 2.5 cm at the tibial
insertion site. Accordingly, the patella, ligament, and tibial bone
block were carefully bisected using the band saw. Each bisected
ligament had a tendon width of at least 1.2 cm. (Note: if the
ligament at the insertion site is less than 2.5 cm, the patella,
ligament, and tibial bone block should not be bisected.) The tibial
tuberosity bone block was trimmed to yield a rectangular appearing
section, at least 1.2 cm wide, 2.8 cm long, and 1.2 cm thick
Anterior-Posterior (A-P). The fashioned tibial tuberosity bone
block should be the same width as the ligament at the insertion
site (at least 1.2 cm).
The graft was then placed in a basin containing a 1:100 dilution of
ALLOWASH.TM. solution for 15 minutes. The graft was placed into an
empty basin and rinsed three times with copious amounts of sterile
water to remove any residual bone cleaning solution. Any sterile
water which accumulated in the basin was discarded.
The entire fashioned graft was placed in a small basin containing
U.S.P. grade 70% isopropyl alcohol for 5 minutes. The graft should
not be left in the isopropyl alcohol for more than 5 minutes, as
this desiccates the tissue. The graft was then placed in a sterile
basin containing an antibiotic solution for 15 minutes. The graft
was then soaked thoroughly by immersing the deposit into a sterile
water bath for 5 minutes to remove any remaining reagents.
Pulsatile lavage was then used to remove any remaining marrow
elements from the bone. The fashioned graft was then placed on
sterile fine mesh gauze and the gauze was trimmed to just beyond
the edges of the graft.
The bisected graft was placed on sterile fine mesh gauze, the gauze
was trimmed to just beyond the edges of the graft and the
graft/gauze combination was placed into a 250 ml bottle.
(Note: If the fashioned graft(s) are to be frozen, the fashioned
graft is cultured for bacterial contamination. One cotton-tipped
applicator is placed into a thiogylocolate broth tube and one into
a trypticase-soy broth tube. The graft material is then ready for
packaging/wrapping and placement in either the freeze dryer or
appropriate freezer.)
TABLE II
__________________________________________________________________________
Patellar Ligament Strength Data Units: kiloPascals unless noted
Tensile Tensile Tensile Force Strain Rate Strength Tensile Force
Strain Rate Donor ID Strength BCS (N) BCS (In/In) BCS NBCS (N) NBCS
(In/In) NBCS
__________________________________________________________________________
95-1119 7636 2290 0.46 6971 2415 0.38 96-0162 5791 2241 0.55 9393
2994 0.53 96-0187 2537 972 0.42 3890 1315 0.45 96-0190 8081 2059
0.52 7054 1966 0.5 96-0204 6676 1903 0.46 6544 1992 0.56 96-0212
3189 862 0.41 1807 524 0.58 96-0269 4796 1748 0.55 6700 2460 0.39
96-0300 5802 2055 0.64 5737 2139 0.52 96-0565 7207 1953 0.53 7206
3674 0.49 96-0591 6024 2002 0.53 6431 2099 0.51 96-0619 7858 2793
0.46 7251 2709 0.45 8409-10135 9077 2922 0.47 8734 3251 0.45
Average 6222.83 1983.33 0.50 5476.50 2294.83 0.48 SD 1969.594
606.873 0.065 2009.296 846.213 0.062 Max/Min -- 2922/972 -- --
3674/524 -- Range -- 1950 -- -- 3150 --
__________________________________________________________________________
Example V
Fascia lata
Any remaining muscle tissue was first removed from the fascia lata.
The fascia lata was then placed with the subcutaneous layer
uppermost, on a clean, blue drape towel. Using blunt dissection
techniques, all of the fat and extraneous soft tissue was removed
from the graft material. The graft was kept moist with isotonic
antibiotic solution to prevent desiccation during processing. Any
torn fibers were removed from the edges of the graft material,
creating a graft of rectangular shape. Care should be taken to
obtain the largest graft possible. (Note that this procedure can
process a single graft or multiple grafts.)
The graft was then placed in a basin containing a 1:100 dilution of
ALLOWASH.TM. solution for 15 minutes. The graft was then placed
into an empty basin and rinsed three times with sterile water to
remove any residual surfactant. Any sterile water which accumulated
in the basin was discarded.
The fashioned graft was then placed in a basin containing 70%
isopropyl alcohol for 5 minutes. Exposure of the graft to alcohol
for more than 5 minutes is avoided, to reduce desiccation of the
tissue. The graft was then placed in a basin containing an
antibiotic solution for 15 minutes.
The graft was then thoroughly soaked by immersion in a sterile
water bath for 5 minutes, to remove any remaining reagents.
Thereafter, the fashioned graft was placed on sterile fine mesh
gauze, and the gauze was trimmed to just beyond the edges of the
graft. The width and length of the graft(s) were then measured to
the nearest tenth of a centimeter.
The graft(s) and gauze were rolled into tubes and placed into a 250
ml glass bottle. In operations were multiple grafts are processed,
this step is repeated until all grafts are bottled.
If the fascia lata is to be preserved in U.S.P. grade 0.9% sodium
chloride for irrigation, the measured deposit is placed into a
sterile 120 ml bottle. Approximately 60 ml of U.S.P. grade 0.9%
sodium chloride for irrigation is poured into the bottle and the
deposit is covered. A stopper is placed in the bottle and pressed
tightly to prevent the saline from leaking. These steps were
repeated for each representative sample deposit.
Then the fascia lata deposit(s) and representative sample pieces
are wrapped according to the procedure for wrapping freeze dried
tissue and transported to the freeze dryer room. The deposits are
placed in a disinfected freeze dryer, the stopper is loosened, and
a vacuum in the dryer is drawn.
TABLE III ______________________________________ Fascia Lata
Tensile Force Strain (In/In) Tensile Force Strain (In/In) Donor ID
(N) BCS BCS (N) NBCS NBCS ______________________________________
96-0162 298 0.54 291 0.47 96-0162 405 0.59 387 0.61 96-0162 170
0.64 576 0.62 96-0162 177 0.4 347 0.54 96-0187 210 0.42 141 0.36
96-0187 211 0.37 171 0.5 96-0187 175 0.31 -- -- 96-0187 147 0.31
168 0.33 96-0190 211 0.4 467 0.24 96-0190 229 0.42 481 0.24 96-0190
156 0.31 253 0.17 96-0190 164 0.22 317 0.22 96-0212 207 0.53 328
0.53 96-0212 456 0.5 304 0.49 96-0212 140 0.42 369 0.41 96-0212 265
0.39 375 0.49 96-0269 244 0.24 355 0.43 96-0269 283 0.23 407 0.37
96-0269 205 0.19 -- -- 96-0269 -- -- 325 0.26 96-0304 283 0.47 222
0.46 96-0304 129 0.37 212 0.3 96-0304 159 0.49 199 0.4 96-0304 194
0.44 159 0.27 95-1415 347 0.43 422 0.68 95-1415 262 0.57 363 0.37
95-1415 412 0.44 355 0.49 95-1415 431 0.48 335 0.45 96-1525 -- --
434 0.34 96-1525 182 0.29 564 0.35 96-1525 114 0.23 237 0.31
96-1525 -- 0.23 326 0.23 AV- 236.759 0.395 329.667 0.398 ERAGE SD
94.322 0.120 112.606 0.130 Max/Min 456/114 -- 576/141 -- Range 342
-- 435 -- ______________________________________ BCS = Bone
Cleaning Solution (0.01X Allowash Solution) NBCS = No Bone Cleaning
Solution -- = no data available (N) Newtons
Results
No significant differences were observed in the tensile strength of
untreated Achilles tendons as compared to tendons treated with a
bone cleaning solution of the present invention. No significant
differences were observed in the tensile strength of fascia lata
treated with bone cleaning solution when compared with untreated
fascia lata.
The tensile strength of patellar ligaments was tested. No
significant differences were observed between bone cleaning
solution treated grafts and untreated grafts.
The tensile strength of fascia lata was tested. No significant
differences were observed between bone cleaning solution treated
grafts and untreated grafts.
The Young's modulus of untreated Achilles tendons and Achilles
tendons treated with bone cleaning solution was measured. No
significant differences were observed between bone cleaning
solution treated grafts and untreated grafts.
The Young's modulus of untreated patellar ligaments and patellar
ligaments treated with bone cleaning solution was measured. No
significant differences were observed between bone cleaning
solution treated grafts and untreated grafts.
The Young's modulus of untreated fascia lata of males and female
and fascia lata of males and female with bone cleaning solution was
measured. No significant differences were observed between bone
cleaning solution treated grafts and untreated grafts.
The type and frequency of patellar ligament failures after
treatment with a present bone cleaning solution and without
treatment with a present bone cleaning solution were found to be as
follows in Table V:
TABLE IV ______________________________________ Failure ALLOWASH
.TM. No ALLOWASH .TM. ______________________________________
Cracked Bone 4 5 Tear at Bone 9 6 Tissue Tear 2 0 Other 0 3
______________________________________
TABLE V ______________________________________ Young's Modulus Data
(Tensile Strength) Units: kiloPascals AVERAGES Donor ID BCS Treated
NBCS Treated ______________________________________ 95-1119 44713
34081 96-0162 34385 23044 96-0187 15369 28065 96-0190 41091 35590
96-0204 27552 12206 96-0212 18413 16501 96-0269 24730 55904 96-0300
29238 25605 96-0565 57603 67653 96-0591 26933 44974 96-0619 44787
37912 B409-10135 61179 35515 GRAND AVERAGE 35499.42 34754.17
______________________________________ BCS = Bone Cleaning Solution
(0.01X Allowash Solution) NBCS = No Bone Cleaning Solution
Example VI
Viral Inactivation Data for the Soft Tissue Treatment by
ALLOWASH.TM. Solution.
This experiment tested the antiviral effect of ALLOWASH.TM.
Solution against HIV-1/LA1 in 3 day PHA stimulated PBM cells from
the Red Cross. RTU media contained HR-IL2 (26.5 Units/ml). Virus
was incubated with varying amounts of ALLOWASH.TM. solution for 5
minutes at room temperature. This experiment was performed in a 24
well plate 4.times.10(5) cells/ml. One ml of supernatant was spun
at 12,000 rpm for 2 hours at 4.degree. C. using the Jouan MR 1822.
10 ml of resuspended sample of RT'd using 75 ml of 4.times.3H-TTP
cocktail. A semi-automated RT was performed on disrupted pellets
using the Packard harvester and direct beta counter. All CPM values
are averages of duplicate RT's.
The results indicated that at less than 0.0025.times. Allowash
solution (cleaning is generally performed using a concentration of
0.01.times. at 0.01 times) 100% of the HIV was inactivated within 5
minutes at room temperature.
TABLE VI ______________________________________ CMP/ % Inhibition
Treatment Concn. CPM ml Comments (corrected)
______________________________________ Blanks 40 82 Average 61
Stdev 30 HIV Std. 1,591 18,910 Virus Titration 2.5 .mu.l 789 7,890
Average 1,049 10,490 Stdev 919 9,190 10 .mu.l 1,402 14,020 1,677
16,770 Average 1,540 15,400 Stdev 194 1,945 UI Controls 240 2,400
364 3,640 196 1,960 Average 224 2,235 Stdev 112 1,119 inf. Control
963 9,630 HIV-1/LA1 1,134 11,340 P-89, 4.17-5.01 1,168 11,680 from
PMB cells 1,957 19,570 5 .mu.l of virus/well Average 1,306 13,060
0.0 Stdev 444 4,436 Virus 5 .mu.l+ 0.025X 109 1,090 110.6 EC.sub.50
= <0.025X 10X Allowash 230 2,300 99.4 5 .mu.l 194 1,940 102.7 65
650 114.6 Average 150 1,495 106.5 Stdev 76 758 105.5 Virus 5 .mu.l+
0.0125X 102 1,020 111.2 EC.sub.50 = 0.0125X 10X Allowash 94 940
112.0 5 .mu.l 211 2,110 101.2 238 2,380 98.7 Average 161 1,610
105.8 Stdev 74 740 Virus 5 .mu.l+ 0.0025X 215 2,150 100.8 EC.sub.50
= <0.0025X 1X Allowash 5 .mu.l 83 830 113.0 80 800 113.2 92 920
112.2 Average 118 1,175 109.8 Stdev 65 652
______________________________________
Although the invention has been described with reference to
particular means, materials and embodiments, it is to be understood
that the invention is not limited to the particulars disclosed and
extends to all equivalents within the scope of the claims.
All the references cited above are incorporated herein in their
entirety by reference.
* * * * *